Directed evolution reprograms botulinum neurotoxins

Proteases are enzymes that cleave other proteins at defined locations. Such enzymes are attractive as therapeutics, but reprogramming them to selectively target a specific substrate is challenging. The natural substrates of botulinum neurotoxin (BoNT) proteases, including the enzyme marketed as Botox, are proteins involved in the formation and release of vesicles in neurons. These enzymes are examples of clinically used proteases that researchers have attempted to repurpose with varying degrees of success. Now researchers have used directed evolution to successfully repurpose enzymes in this class, potentially opening a route to develop new anticancer drugs. David R. Liu of Broad Institute of MIT and Harvard and the Howard Hughes Medical Institute, Min Dong of Harvard Medical School, and coworkers have now used a directed evolution method called phage-assisted continuous evolution (PACE) to reprogram the specificity of BoNT proteases so they target proteins other than their natural substrates (Science 2021, DOI: 10.1126/science.abf5972). The researchers used both positive and negative selection PACE to evolve BoNT proteases that selectively target desired substrates. They evolved three distinct classes of BoNT proteases into variants that selectively cleave each of four protein targets chosen by the researchers, including phosphatase and tensin homolog, a signaling protein that is mutated in many cancers and is unrelated to any known native targets of natural BoNT proteases.